Cobalt-substituted cytochrome P-450cam.

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Enzymology

Background:

• Cytochrome P-450cam is a crucial enzyme involved in substrate hydroxylation.
• Understanding the coordination environment of the heme iron is essential for elucidating enzyme mechanisms.
• Cobalt-substituted porphyrins serve as valuable analogues for studying heme proteins.

Purpose of the Study:

• To create a cobalt-substituted P-450cam analogue.
• To characterize the spectral and enzymatic properties of the cobalt analogue.
• To investigate the axial ligand coordination in both the cobalt analogue and native P-450cam.

Main Methods:

• Reconstitution of apo-cytochrome P-450cam with cobalt protoporphyrin.
• Spectroscopic analysis including optical and electron paramagnetic resonance (EPR).
• Enzymatic assays to assess substrate binding and hydroxylation activity.

Main Results:

• The cobalt P-450cam analogue exhibited faithful spectral, ligand-binding, and enzymatic characteristics compared to the native enzyme.
• Unique Soret "hyper" spectra were observed for thiol and cyanide complexes of the cobalt analogue.
• Substrate-induced spectral changes and limited hydroxylation activity were retained.
• EPR studies indicated a thiolate axial ligand in both reduced and oxygenated cobaltous P-450cam.

Conclusions:

• Cobalt P-450cam serves as a valid analogue for studying the native enzyme.
• The presence of a thiolate axial ligand is strongly suggested in the cobalt P-450cam.
• These findings imply that native ferrous and oxygenated P-450cam also retain a thiolate axial ligand, providing insights into the enzyme's active site structure and mechanism.